I have been carrying out Campylobacter flaRFLP for about a month. After I have amplified the target (flaA) by PCR, I cut the target with DdeI enzyme (Fermentas) for 8 hours according to the procedure suggested by Fermentas. I am using % 4 Nusieve GTG agarose and 100 bp ladder between 100-1000 bp during electrophoresis and rUn the digests at 90 V for 90 minutes. After staining with etidium broimde, the bands in the lower base pairs are faint. They are not as strong as the bands in the higher base pairs.

Wait a sec. So you amplify your GOI and then cut it with RE for what exactly? Just at the ends to make sticky ends for consequent cloning? And I guess you use your PCR reaction without any purification?

I do not have any purpose of cloning.My aim is to type 300 Campylobacter strains molecularly and look for similarity between isolates; in short my aim is molcular typing. I am using RFLP just for that. Not for cloning or for something genetical method.